Cytochrome P450 2E1 (CYP2E1) is a heme-containing monooxygenase that catalyzes the oxidation of a number of hepatotoxins and procarcinogens including ethanol, acetaminophen, nitrosamines, and carbon tetrachloride, among many others. CYP2E1 is highly inducible in human livers by alcohol. Upregulation of CYP2E1 by alcohol enhances hepatotoxicity and increases the risk of developing cancer in alcoholics. It is thought that CYP2E1 plays an important role in the pathogenesis of alcohol-induced liver injury due to CYP2E1-mediated oxidative stress and lipid peroxidation. Inhibition of CYP2E1 activity has been shown to minimize the toxicity of alcohol. However, existing CYP2E1 inhibitors cannot be used in vivo because of their promiscuity and toxicity. There is a need to develop specific and non-toxic CYP2E1 inhibitors that can be used both for basic research to investigate the specific role of CYP2E1 in alcohol-related diseases and for use in the clinic to treat and prevent alcoholism. I hypothesize that potent and specific mechanism-based inhibitors of CYP2E1 can be identified from small ligand libraries through virtual high throughput screening (vHTS). In this proposal, I plan to test this hypothesis with two specific aims: 1) to screen approximately 55,000 acetylenic compounds in small ligand libraries using dual selection criteria and 2) to analyze the potency and specificity of the vHTS hits by biochemical assays. The outcome of this proposal will yield approximately a dozen lead inhibitors for CYP2E1 that can be used to accelerate the development of therapeutic agents.